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Comparison of Cathepsin B Activity

and the Ratio of Cathepsin B to Cysteine Peptidase

Inhibitor in Human Colorectal Cancer Tissue

Porównanie aktywności katepsyny B

i indeksu katepsyna B/inhibitory peptydaz cysteinowych

w ludzkiej tkance nowotworów okrężniczo−odbytniczych

1 _{Department of Pathophysiology, Wrocław Medical Unversity, Poland}

2 _{Department of Pharmaceutical Biochemistry, Wrocław Medical Unversity, Poland} 3 _{Faculty of Public Health, Wrocław Medical Unversity, Poland}

4 _{Regional Specialist Hospital, Wrocław, Poland}

Adv Clin Exp Med 2009, 18, 1, 41–45 ISSN 1230−025X

ORIGINAL PAPERS

Abstract

Background.Cathepsin B (CB), which is inhibited by cysteine peptidase inhibitors (CPIs), plays an important role in the process of cancer invasion and metastasis.

Objectives. The aim of this study was to compare CB and low−molecular−weight CPI activities in the extracts of human colorectal carcinoma tissues from the center of the tumor and the tumor−free area 2 cm and 5 cm from the tumor border in order to examine their significance in this type of cancer.

Material and Methods.CB activity was measured using a flurogenic substrate and CPI using a colorimetric sub− strate as anipapain activity. The CB:CPI ratio was also calculated.

Results.The activities were significantly higher in the tumor tissue extracts than in the controls (p≤0.0001). CB was elevated 26−fold and CPI 2−fold. Only cathepsin B activity decreased significantly with distance from the tumor border (p≤0.0001). CB and CPI activities as well as the CB:CPI ratio increased with the stage of tumor tis− sue differentiation grade.

Conclusion.The results provided convincing evidence that the distribution of CB and CPI may be important for colorectal cancer invasion. CB activity of cancerous tissue extracts can be an additional marker of tissue differen− tiation grade. For patients with marked elevation of CB activity in the cancer tissue, relatively extensive resection may be necessary (Adv Clin Exp Med 2009, 18, 1, 41–45).

Key words:colorectal cancer, cathepsin B activity, cysteine peptidase inhibitors, cystatins.

Streszczenie

Wprowadzenie.Katepsyna B (CB), hamowana przez inhibitory peptydaz cysteinowych (CPI), odgrywa istotną ro− lę zarówno w powstawaniu, jak i inwazji procesu nowotwowego.

Cel pracy.Porównanie i poznanie istotności ekspresji CB oraz CPI w ekstraktach ludzkich nowotworów okrężni− czo−odbytniczych z tkanek pobranych ze środka guza (0) oraz w odległości 2 i 5 cm.

Materiał i metody. Aktywność CB oznaczano metodą fluorometryczną, a aktywność CPI metodą kolorymetrycz− ną jako aktywność antypapainową. Obliczano również indeks CB/CPI.

Wyniki. Oznaczane aktywności były istotnie wyższe w ekstraktach tkanek nowotworowych w stosunku do grupy kontrolnej (p < 0,001). CB rosła 26−krotnie, a CPI 2−krotnie. Jedynie aktywność CB malała istotnie statystycznie (p < 0.001) wraz z odległością od środka guza. Wszystkie oznaczane wskaźniki rosły wraz ze stopniem zróżnico− wania guza, ale różnice nie miały charakteru istotnie statystycznego.

Wnioski. Rezultaty pracy wskazują, że dystrybucja ekspresji CB i CPI jest istotna dla inwazji nowotworów okręż− niczo−odbytniczych. Oznaczanie aktywności CB może być pomocne w określeniu stopnia zróżnicowania tkanek guza. U chorych ze znacznym wzrostem aktywności CB w ekstraktach tkankowych byłaby wskazana jak najwięk− sza resekcja guza (Adv Clin Exp Med 2009, 18, 1, 41–45).

Słowa kluczowe:rak okrężniczo−odbytniczy, aktywność katepsyny B, inhibitory peptydaz cysteinowych, cystatyny.

Colorectal cancers (CRC) are a significant cause of morbidity and mortality [1]. Numerous investiga− tions have shown that proteolytic enzymes, such as ly− sosomal cysteine peptidases (e.g. cathepsins B and L), are involved in the growth, invasion, metastasis, and angiogenesis of cancer [2–4]. Of the cysteine cathepsins, the most investigated is cathepsin B (CB). Increased CB expression, measured as enzyme activity or antigen concentration (ELISA) as well as by proteomic screens, has been observed in colorectal neoplastic tissues compared with nor− mal mucosa [4–6]. The expression and activity of CB and cathepsin L (CL) antigen is associated with early CRC progression. Survival of patients with potentially curable disease was inversely related to both CB and CL activities in tumor tissue [7]. High activities of these enzymes were found particularly in cells at the invasion front, namely in the “tumor budding” region, which is generally characterized by a high expression of proteases [8], which sug− gested a more aggressive tumor phenotype [9]. It has been also shown that overexpression of CB in can− cer cells of colon adenocarcinoma correlates with the intensity of angiogenesis [10].

The activities of cysteine peptidases are con− trolled within cells by the cytosolic inhibitors stefin A and B [11]. In colon cancers expressing CB, the surrounding tissue also demonstrated increased amounts of cystatin C, a secreted inhibitor [12]. Alteration of the balance between cysteine peptidas− es and their endogenous inhibitors has been postu− lated to contribute to malignant progression and may

participate in the modulation of the invasive pheno− type of tumors [4, 11, 13]. Recombinant cystatin C, characterized by high inhibitory activity against CB, effectively and dose−dependently retards the growth and invasiveness of human colon carcinoma [14].

Based on the hypothesis that an imbalance between cysteine peptidases and their inhibitors leads to cancer invasion, the current study investi− gated the activity of CB and the antipapain activi− ty of cysteine peptidase inhibitors (CPIs) and cal− culated CB:CPI ratio in extracts of human col− orectal carcinoma tissues obtained at different distances from the tumor border. Antipapain activ− ity reflects the expression of all low−molecular− weight inhibitors (cystatin C and stefins A and B).

Material and Methods

Thirty−eight tissue samples of colorectal carci− nomas and of 30 normal mucosa were obtained from patients undergoing surgery at the Silesian Center of Oncology, Wrocław, Poland. The patients were informed of the aim of this investi− gation and gave their permission to participate in the study. The ages of the patients ranged from 38 to 68 years. These tissue samples were classified according to the routine histological hematoxylin and eosin (H&E) staining as colorectal adenocar− cinoma and carcinoma (Table 1). Tumor−node− metastasis (TNM) staging was done in accordance with the American Joint Committee on Cancer

Table 1.Cathepsin B activity and the CB:CPI ratio in normal and colorectal tissue extracts with cancer of different histol− ogy type and cell differentiation stage shown as mean ±SD

Tabela 1.Aktywność katepsyny B i indeks CB:CPI w tkance prawidłowej i w ekstraktach nowotworów okrężniczo−odbyt− niczych różnego typu histologicznego i stopnia różnicowania komórek – średnia ±SD

Histology type Cathepsin B CPI – U/mg of protein CB:CPI Ratio

Tissue differentiation grade – U/mg of protein (CPI – U/mg białka) (Wskaźnik CB:CPI)

(No of patients) (Katepsyna B

(Typ histologiczny – U/mg białka) (Stopień zróżnicowania tkanki)

(Liczba pacjentów)

Normal tissue (30) 6.6 ±5.8 0.2 ± 0.1 33

Carcinomas

G1(5) 22.8 ±8.7 0.3 ±0.1 76

G2(10) 103.2 ±95.6 0.6 ±0.3 172

G3(3) 286.9 ±158.4 0.9 ±0.4 319

Adenocarcinoma 457.5 ±275.2 0.5 ±0.2 915

G2(7)

Invasive adenocarcinoma 621.4 ±345.7 0.8 ±0.3 777

G3(13)

All values for the cancer tissues were significantly higher (p < 0.001) than for the normal tissue. In both histopathological types the differences in CB and CPI between subgroups were not statistically significant.

(AJCC). Five carcinoma tissues (13%) were well− differentiated (grade G1), ten (26%) moderately differentiated (grade G2), and three (8%) poorly differentiated (grade G3). Seven tubular adenomas with high−grade dysplasia/carcinoma in situ (18%) of grade G2 and thirteen invasive adenocarcino− mas (34%) of grade G3 were also collected. Moreover control mucosa samples taken from nor− mal tissue located at least 10 cm from the tumor site were analyzed.

Immediately after surgery a pathologist select− ed the tissues and washed them of blood with 0.9% NaCl. For CB determinations the frozen tissues (0.5 g) were homogenized with 2.5 ml of buffer (20 mM Tris−3 mM EDTA−1 mM dithiothreitol, pH 7.6) at 4°C. The homogenates were centrifuged for 6 min at 13,000 × gat 4°C. To determine the antipapain activity of the low−molecular−weight cysteine pro− teinase inhibitors (CPIs), tissue homogenates were prepared according to Lenney et al. [15]. Dissociation of the cysteine peptidase inhibitor complexes was done by heating the homogenates at 100°C for 5 min and subsequent centrifugation. Appropriate extracts (supernatants) were used for determining CB and antipapain activity.

CB activity was measured according to Barrett et al. [16] using the fluorogenic synthetic substrate benzyloxycarbonyl−arginyl−arginyl−AMC (Z−Arg− Arg−AMC). Fluorescence of the released 7−amino− methylcoumarin (7−AMC) was measured by a spectrofluorimeter (Perkin−Elmer LS−3B) at 370 nm excitation and 440 nm emission wavelengths. One unit of enzyme activity was defined as 1 nM of 7−AMC released per minute. Antipapain activi− ty of heat−stable low−molecular−weight inhibitors was assayed using N−α−benzylarginine−β−naph− thylamide as the substrate according to Barrett [17]. One unit of CPI activity was defined as the amount required to inhibit 1 unit of papain activi− ty, which was previously determined by active site titration with E−64 [18]. The protein concentration was determined by the Bradford method using bovine serum albumin as the standard [19].

The biochemical analysis results are presented as the mean values ±SD. Analyses of the differ− ences between the results obtained in tumor and control tissue homogenates were performed with the Wilcoxon signed rank test; a level of probabil− ity lower than 0.05 was taken as significant.

Results

Figure 1 presents the median values of CB and CPI activity and the CB:CPI ratios in the extracts of malignant and nonmalignant colorectal tissues. All the parameters were significantly higher in the

control grupa kontrolna

0 2 5

0 500 1000 1500

activity [U/mg protein] aktywnoœæ [J/mg bia³ka]

distance from tumor border [cm] odleg³oœæ od obrze¿a guza [cm]

Cathepsin B

* *

* * *

control grupa kontrolna

0 2 5

0.00 0.25 0.50 0.75

CPI activity [U/mg protein]

aktywnoœæ [J/mg bia³ka]

distance from tumor border [cm] odleg³oœæ od obrze¿a guza [cm]

Fig. 1.Cathepsin B and antipapain activity of CPI and the CB:CPI activity ratio in colorectal cancer tissue extracts. The significance of the differences (p) in me− dian values of tumor and control tissue were calcula− ted by Wilcoxon matched pair signed−rank test. * p< 0.001 compared with control tissue

** p< 0.001 compared with 2 or 5 cm from the tumor border

Ryc. 1. Aktywność katepsyny B i antypapainowej CPI i wskaźnik CB:CPI w ekstraktach nowotworów okręż− niczo−odbytniczych. Istotność różnic (p) mediany war− tości dotyczących guza i grupy kontrolnej obliczono za pomocą testu Wilcoxona.

* p< 0,001 w porównaniu z grupą kontrolną

** p< 0,001 w porównaniu z tkanką pobraną w odle− głości 2 lub 5 cm od obrzeża guza

control grupa kontrolna

0 2 5

0 1000 2000 3000 4000

Cathepsin B/CPI

* *

* * * ratio

indeks

tumor tissues than in the control tissues (p≤0.0001). CB was elevated 26−fold, CPI 2−fold, and CB:CPI 10−fold. Both CB activity and the CB:CPI ratio were significantly lower at distances of 2 and 5 cm from the tumor border (p≤0.0001). The differences in antipapain activity were not sta− tistically significant.

The analysis of the results with respect to his− tology and stage of cell differentiation are shown in Table 1. It is evident that CB and CPI activity increase with the stage of disease. In carcinomas, CB activity rose from 22.8 ±8.7 in G1 to 103.2 ± 95.6 in G2 to 286.9 ±158.4 U/mg of protein in G3. In adenocarcinomas, CB activity was even greater than in carcinomas, with 457.5 ± 275 in G2 and 621.4 ±345.7 U/mg of protein in G3. The antipa− pain activity of the cystatins in carcinomas was 0.3

±0.1 in G1, 0.6 ±0.3 in G2, and 0.9 ±0.4 U/mg of protein in G3. The differences between the groups were not statistically significant. The dif− ferences between the inhibitory activity obtained for G2 adnocarcinomas and invasive G3 adenocar− cinoma were not statistically significant, in con− trast to the CB:CPI ratio (p≤0.0001).

Discussion

Cysteine peptidases (e.g. CB) of various tumor cells, including colorectal carcinoma, were shown to be involved in cancer progression and metasta− sis [2–5]. It is now evident that, depending on their location, they can be involved in two apparently opposing mechanisms: tumor invasion and apop− totic regression. Enzymes that are secreted or asso− ciated with the plasma membrane participate in tumor invasion through proteolytic cascade activa− tion, ECM degradation, and inactivation of cell− adhesion proteins. In addition, intracellular prote− olysis contributes to progression by degradation of endocytosed collagen in lysosomes. On the other hand, cytosol−translocated cysteine cathepsins trigger apoptosis via BH3−interacting death domain cleavage followed by cytochrom c release from mitochondria. This cascade activated down− stream executioner caspases, resulting in tumor cell death [20].

In the present study, CB was elevated (26−fold) in colorectal carcinoma tissue extracts compared with normal mucosa. CB activity increased with the tissue differentiation grade, from G1to G3,and was higher in adenomas than in carcinomas. Such results are in accordance with other observations [2, 5, 21]. The present study also searched for CB activity in extracts of tissue taken 2 and 5 cm from the tumor center and observed decreased activity with distance, but the values were still significant− ly higher than in the control tissues (p≤0.0001). Such results might provide some suggestion in designating the extent of a CRC operation.

In the present study, CPI antipapain activity was also found to be increased compared with the control tissue extracts (p≤0.0005), but the differ− ences in the distance from the tumor border and between histology or tissue differentiation grade were not statistically significant. The observed changes indicate a shift in the CB−CPT balance, which may enhance carcinogenesis. Others found altered mRNA expression of cystatin C in 55% of examined colon cancers [22]. Interestingly, they also established cystatin C as a novel TGF−β receptor antagonist.

The present authors’ previous study demon− strated immunochistochemically, using anti−chick− en cystatin antibodies, higher expression of cys− tatin C in CRC tissue than in controls. The inhibitor was found in the cytoplasm and on the cell surface in CBC tissue [23]. Altered extracellu− lar levels of stefin A and B and cystatin C corre− lated significantly with a high risk of adverse out− come in cancer patients [24]. Determination of cystatin C−cathepsin B complex concentrations in serum has been suggested as a prognostic indica− tor for cancers, for example CRC [25].

The present results provide convincing evidence that an imbalance of CB and its tissue inhibitors may contribute to CRC invasion. Determining CB activ− ity in tissue extracts seems to be more suitable than CPI antipapain activity in the monitoring of CRC progression and could be an additional marker of tis− sue differentiation grade in this cancer.

References

[1] Denlinger CS, Cohen SJ:Progress in the development of prognostic and predictive markers for gastrointestinal malignancies. Curr Treat Options Oncol 2007, 8, 339–351.

[2] Herszenyi L, Plebani M, Carraro P, De Paoli M, Roveroni G, Cardin R, Foschia F, Tylassay Z, Naccarato R, Farinati F:Proteases in gastrointestinal neoplastic diseases. Clin Chim Acta 2000, 291, 171–187.

[3] Siewiński M, Saleh Y, Ziółkowski P: Cysteine peptidases in health and diseases. Folia Med Cracov 2003, 44, 169–178.

[5] Berdowska I:Cysteine proteases as disease markers. Clin Chim Acta 2004, 342, 41–69.

[6] McKerrow JH, Bhargava V, Hansell E, Huling S, Kuwahara T, Matley M, Coussens L, Warren R:A func− tional proteomics screen of proteases in colorectal carcinoma. Mol Med 2000, 6, 450–460.

[7] Troy AM, Sheahan K, Mulcahy HE, Duffy MJ, Hyland JM, O Donoghue DP:Expression of cathepsin B and L antigen and activity is associated with early colorectal cancer progression. Eur J Cancer 2004; 40, 1610–1616.

[8] Guzińska−Ustymowicz K, Zalewski B, Kasacka B, Piotrowski Z, Skrzydlewska E: Activity of cathepsin B and D in colorectal cancer: relationship with tumour budding. Anticancer Res 2004, 24, 2847–2851.

[9] Guzińska−Ustymowicz K.: MMP−9 and cathepsin B expression in tumor budding as an indicator of a more aggressive phenotype of colorectal cancer (CRC). Anticancer Res 2006, 26, 1589–1594.

[10] Kruszewski WJ, Rzepko R, Wojtacki J, Skokowski J, Kopacz A, Jaskiewicz K, Drucis K: Overexpression of cathepsin B correlates with angiogenesis in colon adenocarcinoma. Neoplasma 2004, 51, 38–43.

[11] Keppler D: Towards novel anti−cancer strategies based on cystatin function. Cancer Lett 2006, 235, 159–176.

[12] Hirai K, Yokoyama M, Asano G, Tanaka S: Expression of cathepsin B and cystatin C in human colorectal can− cer. Hum Pathol 1999, 30, 680.

[13] Corticchiato O, Cajot JF, Abrahamson M, Chan SJ, Keppler D, Sordat B: Cystatin C and cathepsin B in human colon carcinoma: expression by cell lines and matrix degradation. Int J Cancer 1992, 52, 645–652.

[14] Ogawa M, Jing H, Kitts DD, Nakai S, Nakamura S: In vitro anti−cancer activities in Caco−2 and HCT−116 cells of recombinant cystatin C prepared by Pichia expression system. J Med Food 2003, 6, 317–333.

[15] Lenney JF, Tolan JR, Sugai WJ, Lee AG: Thermostable endogenous inhibitors of cathepsin B and H. Eur J Biochem 1979, 101, 153–161.

[16] Barrett AJ:Fluorimetric assays for cathepsin B and H with methylocumarylamide substrates. Biochem J 1980, 50, 303–311.

[17] Barrett AJ, Kirschke H:Cathepsin B, cathepsin H, and cathepsin L. Methods Enzymol 1981, 80, 535–561.

[18] Turk D, Guncar G, Podobnik M, Turk B: Revised definition of substrate binding sites of papain−like cysteine proteases. Biol Chem 1998, 379, 137–147.

[19] Bradford MM: A rapid and sensitive method for the quantitation of microgram quantities of protein utilising the principle of protein−dye binding. Anal Biochem 1976, 72, 248–254

[20] Vasiljeva O, Turk B: Dual contrasting roles of cysteine cathepsins in cancer progression: Apoptosis versus tumour invasion. Biochimie 2008, 90, 380–386.

[21] Talieri M, Papadopoulou S, Scorilas A, Xynopoulos D, Arnogianaki N, Plataniotis G, Yotis J, Agnati N:

Cathepsin B and cathepsin D expression in progression of colorectal adenoma to carcinoma. Cancer Lett 2004, 205, 97–106.

[22] Sokal J P, Schlemann WP: Cystatin C antagonizes transforming growth factor βsignaling in normal and cancer cells. Molec Cancer Res 2004, 2, 183−195.

[23] Saleh Y, Sebzda T, Warwas M, Kopeć W, Ziółkowska J, Siewiński M:Expression of cystatin C in clinical human colorectal cancer tissues. J Exp Ther Oncol 2005, 5, 49–53.

[24] Kos J, Werle B, Lah T, Brunner N:Cysteine proteinases and their inhibitors in extracellular fluids: markers for diagnosis and prognosis in cancer. Int J Biol Markers 2000, 15, 84–89.

[25] Kos J, Krasovec M, Cimerman N, Nielsen HJ, Christensen IJ, Brunner N: Cysteine proteinase inhibitors stefin A, stefin B, and cystatin C in sera from patients with colorectal cancer: relation to prognosis. Clin Cancer Res 2000, 6, 505–511.

Address for correspondence:

Tadeusz Sebzda

Department of Pathophisiology Wrocław Medical University Marcinkowskiego 1

50−368 Wrocław Poland

Tel.: +48 71 784 00 60

Conflict of interest: None declared Recived: 31.10.2008